Investigation of the molecular basis of the regulation of human prothrombin activation by the blood coagulation protein cofactor, factor Va and the control of cofactor activity by the anticoagulant, protein C system are proposed with the long term goal of defining the molecular mechanisms of factor Va functions. For prothrombin activation, the current hypotheses focus on the unknown structural basis of factor Va-prothrombin interactions and their roles in factor Va activity. Specific binding of factor Va with prothrombin, the two activation intermediates, and the reaction products, including thrombin, are postulated to be mediated by the prothrombin fragment 2 domain and/or a site in the catalytic domain that binds the hirudin anticoagulant dodecapeptide. Modulation of the affinities and/or mode of factor Va binding of prothrombin activation species that accompanies the activation bond cleavage reactions is hypothesized to be an essential molecular mechanism of factor Va regulation of the rate and pathway of prothrombin activation. In a related process, interdependent interactions among activated protein C (APC), factor Va, factor Xa, protein S, calcium, and phospholipid membranes are thought to regulate factor Va activity through assembly-directed inactivation of the cofactor by APC proteolysis. The rate of factor Va inactivation is hypothesized to be determined by assembly of an APC-factor Va, cofactor-inactivation complex, enhanced by interactions of protein S that promote complex assembly, and opposed by competition with the formation of procoagulant factor Va complexes through competitive binding of factor Xa and APC to factor Va. Biochemical and biophysical studies employing fluorescence and affinity chromatography techniques to quantitate the binding interactions, in conjunction with kinetic studies of the proteolytic reactions in model systems are proposed to evaluate the hypotheses. Specific aims are: (1) Quantitative characterization of binding of prethrombin 1 and its activation products to factor Va and determination of the roles of binding sites for the hirudin peptide and fragment 2. (2) Elucidation of the roles of factor Va interactions with prethrombin 1 activation species in the mechanism of factor Va activity. (3) Quantitative characterization of binding interactions in assembly of the APC-factor Va, cofactor- inactivation complex and regulation of assembly by factor Xa and protein S. (4) Definition of the mechanism of control of factor Va activity by APC inactivation of the cofactor and the molecular basis of regulation by factor Xa and protein S. The results of the proposed investigation are expected to have significance in furthering the understanding of the molecular mechanism of the normal hemostatic response, and the mechanisms of thrombotic cardiovascular disease. Information derived from these studies may be valuable in developing new diagnostic methods for assessing the risk of thrombosis, may enable improved therapy, and may provide the basis for the design of new anticoagulant drugs which act through the natural regulatory mechanisms.